The present invention relates to compositions and a method for treating a patient having a tumor. More specifically, the present invention relates to the treatment of such patients with an effective amount of a selenophene derivative.
The control and cure of cancer represents one of our most challenging health problems. The treatment of cancer can be approached by several modes of therapy including surgery, radiation, chemotherapy or a combination of any of these treatments. Chemotherapy continues to be an indispensable therapy for inoperable or metastatic forms of the disease.
The selection of natural compounds, or the synthesis of new compounds having effective anticancer activity is complicated by the still limited knowledge of cancer cell biology and biochemistry. Therefore, development of new effective anti-tumor agents will remain heavily dependent on screening compounds to discover novel compounds having cytotoxic activity. Preferably, such compounds exhibit enhanced cytotoxicity against tumor cells relative to their cytotoxicity to normal cells.
The success of novel antitumor drug development programs is dependent on the initial identification of antitumor agents. Thus the discovery of antitumor agents requires the systematic screening of a large number of natural products and synthetic compounds.
The mouse L1210 leukemia cell line was initially the preferred model system used for screening natural compounds for antitumor activity. However, the P388 murine leukemia system was found to be more sensitive and predictive than L1210 leukemia system, and has been used as primary screen during the past decade. Systematic screening for compounds exhibiting toxicity to these two leukemia cell lines has resulted in the isolation of a large number of active natural products. However, the anticancer activities of these compounds were predominantly in leukemia, lymphoma and a few rare tumors. Low clinical efficacy, or the lack of clinical efficacy of known chemotherapeutics against slower growing solid tumors, is a serious concern.
It has been recognized that the use of a single antileukemia screening system could bias the end results and lead to the isolation of compounds only active in the treatment of fast growing tumors. In addition, the use of a single antileukemia screening system may not detect novel compounds with high specificities for particular cell lines. It is also likely that many novel compounds with possible anti-tumor activity have remained undetected by the less sensitive in vivo models due to the low concentrations at which many active natural products occur
Considering the diversity of tumors in terms of cell type, morphology, growth rate and other cellular characteristics, the U.S. National Cancer Institute (NCI) has developed a xe2x80x9cdisease-orientedxe2x80x9d approach to antitumor activity screening (M. R. Boyd, in xe2x80x9cPrinciple of Practice of Oncologyxe2x80x9d J. T. Devita, S. Hellman, S. A. Rosenberg (Eds.) Vol. 3, PPO Update, No. 10, 1989). This in vitro prescreening system is based on the measurement of antitumor cytotoxicity against human tumor cell line panels consisting of approximately 60 cell lines of major human tumors (including leukemia and slower growing tumor cells such as lung, colon, breast, skin, kidney, etc.). The most important advantage of the new in vitro screening panels is the opportunity to identify compounds that are selectively more cytotoxic to cells of slowly growing solid tumors than to rapidly growing leukemia cells.
The cytotoxicity profile of the NCI human tumor cell panels displays the tumor specificity of a given compound, however the assay does not assess the toxicity of that compound to normal human cells. Accordingly a second bioassay is utilized to measure the selective cytotoxicity against certain types of tumor cells verses normal human cells.
The growth and differentiation of cells are regulated by signaling cascades induced by various mitogenic proteins (J. Kuijan and B. L. Taylor, xe2x80x9cSignal Transduction,xe2x80x9d Academic Press, New York, N.Y. 1994) that often are encoded by proto-oncogenes. The overexpression, amplification or mutation of the oncoprotein is critically involved in the initiation, progression and metastasis of malignant cells (R. A. Weinberg, xe2x80x9cOncogenes and the Molecular Origins of Cancer,xe2x80x9d Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989). Many oncoproteins alter normal cellular growth regulation by modulating the intracellular signaling pathways from the membrane to the nucleus. Therefore, cancer may be considered as a disease of cellular signal transduction, which presents a novel approach for anticancer therapy. One of the critical enzymes involved in the oncoprotein signal transduction is protein kinase C (U. Nishizuka, Nature, 308, 693, 1984 and Science, 233, 305, 1986). Thus, the determination of a compound""s ability to inhibit protein kinase C activity has become a good prognostic for discovering novel anticancer agents (A. Basu, Pharmac Ther, 59, 257, 1993). Furthermore it is anticipated that the selenophene compounds will demonstrate selectivity for certain class members of protein kinases, including protein kinage C. Inhibition of a specific classes of protein kinases will allow the treatment of other diseases associated with defects in signaling transduction.
Selenophenes are selenium containing heterocyclic compounds that are analogs of naturally occurring thiophene, furan and pyrrole compounds. Selenophenes have been found to be effective antitumor agents, and exhibit enhanced cytotoxicity against slow growing tumor cells, selective cytotoxicity against human renal, ovarian tumor cells, and skin tumor cells; and exhibit inhibition of protein kinase C.
In accordance with this invention there is provided a method for the treatment of cancer which utilizes selenophene compounds of the formula I: 
wherein R1 and R2 are independently selected from the group consisting of; 
H, CHO, CH2OH and CH2NH2;
X and Y are independently selected from the group consisting of Se, S, O, NCH3 and NH;
R3, R4, R5 and R6 are independently selected from the group consisting of H, CHO, CH2OH and CH2NH2; cyclodextrin complexes of such compounds; and when R3, R4, R5 or R6 is CH2NH2, the pharmaceutically acceptable salt of the compound represented thereby.
Further in accordance with this invention there are provided novel cytotoxic compounds of the above formula and chemotherapeutic pharmaceutical compositions containing said compounds in anti-tumor effective amounts.
Additional objects, features, and advantages of the invention will become apparent to those skilled in the art upon consideration of the following detailed description of preferred embodiments exemplifying the best mode of the invention as presently perceived.
The present invention is directed to selenophene compounds, their pharmaceutical compositions and methods utilizing such compounds/compositions for treating patients having tumor. The selenophene compounds are effective antitumor agents against slow growing tumors, and generally have been found to exhibit high selective cytotoxicity for individual tumor cell lines.
The present selenophene compounds are readily prepared using art-recognized chemical synthesis procedures as exemplified in Example 1 and Examples 3-8. This invention is further envisioned from the chemical concept on the basis of a coherent design as shown in scheme 3 in Example 2. This chemical concept provides the foundation for conceiving the preparation and utility of numerous xe2x80x9chybridxe2x80x9d selenophene compounds containing other related five-membered heterocycles, such as furan, thiophene and pyrrole, and their analogs. Moreover, the practice of this chemical concept is substantiated by Example 2 and Examples 9-33. The anticancer utility of these hybrid selenophene compounds is manifested by (a) selective cytotoxicity for human renal carcinoma cells in comparison to normal human renal cells (Table 1), (b) antitumor cytotoxicity against a variety of human tumor cells (Example 53), (c) in vivo antitumor activity against human lung tumor (Example 54) and (d) inhibition of protein kinase C activity (Table 2).
In corroboration with the above chemical concept, a versatile, alternative synthetic design is further conceived for the preparation of relevant xe2x80x9chybridxe2x80x9d selenophene compounds as in the scheme shown in Example 34. The practice of this synthetic design is supported by Examples 34-50. The anticancer utility of these hydrid selenophene compounds is indicated by (a) selective cytotoxicity for human renal carcinoma cells in comparison to normal human renal cells (Table 1), (b) antitumor cytotoxicity against a variety of human tumor cells (Example 53), (c) in vivo antitumor activity against human lung tumor (Example 54) and (d) inhibition of protein kinase C (Table 2).
The compounds of the present invention are selenophene compounds of the formula I: 
wherein R1 and R2 are independently selected from the group consisting of; 
H, CHO, CH2OH and CH2NH2;
X and Y are independently selected from the group consisting of Se, S, O and NR;
R is H or C1-C7 alkyl;
R3 R4, R5, and R6 are independently selected from the group consisting of nitro, amino, alkoxy, cyano, chloro, bromo, iodo, C1-C7 alkyl or haloalkyl, C1-C7 alkenyl or haloalkenyl, C1-C4 alkanoyloxy methyl, CH2OR7, COR8, CH2NR9R10, CH(OR7)R11, CHxe2x95x90CR12R13, CHxe2x95x90NR14, CH2SC(NH)NH2 and Cxe2x89xa1CR15 wherein
R7 is H, CO(CH2)2CO2H, (CH2)2OCH3, C1-C4 alkyl or COC1-C17 alkyl;
R8 is H or C1-C7 alkyl;
R9 and R10 are independently H, CN, C1-C4 alkyl, or mono- or di-hydroxyC2-C4 alkyl;
R11 is C1-C7 alkyl, or C1-C7 alkenyl;
R12 and R13 are independently H, C1-C7 alkyl, COOR8, CN, CH(OR7)COOR8, Br, CO-thienyl, COC6H4OH(p);
R14 is NHR7 or OR8;
R15 is COOR8, CH(OR7)CH2OR16 or CH(OCOC1-C4 alkyl)CH2OR8;
R16 is H, COCH2CH2CO2H, or COC1-C7 alkyl; cyclodextrin complexes of such compound and when R3, R4, R5 or R6 is CH2NR6R7, the pharmaceutically acceptable salt of the compound represented thereby.
In one preferred embodiment of this invention there is provided anti-tumor selenophenes of the above formula I,
wherein R2 is 
X and Y are independently selected from the group consisting of S, Se and NH;
R1, R3, and R6 are H; and
R5 is selected from the group consisting of CHO or CH2OH; and cyclodextrin complexes of such compounds. These compounds have been demonstrated to exhibit cytotoxic selectivity against transformed human cells (See Table 1).
In another preferred embodiment of this invention there is provided anti-tumor selenophenes of the above formula I wherein R1 is 
X and Y are independently selected from the group consisting of S, Se and NH;
R2, R3, and R6 are H;
R5 is selected from the group consisting of CHO or CH2OH; and cyclodextrin complexes of such compounds.
Other preferred compounds in accordance with this invention are selenophenes of formula I: 
wherein R1 and R2 are independently selected from the group consisting of; 
H, CHO, CH2OH and CH2NH2;
X and Y are independently selected from the group consisting of Se, S, O, NCH3, and NH;
R3, R4, R5 and R6 are independently selected from the group consisting of H, CHO, CH2OH and CH2NH2; cyclodextrin complexes of such compounds; and when R2 or R3is CH2NH2, the pharmaceutically acceptable salt of the compound represented thereby; with the proviso, that when R2 is 
In accordance with another embodiment of the present invention novel intermediates of Formula II are also provided: 
wherein W is selected from the group consisting of N(CH3)2 and 
and X and Y are independently selected from the group consisting of Se, S, O, NCH3 and NH.
One aspect of the present invention is a method of preparing the compounds of Formula I through an intermediate a compound of the formula 
in accordance with the general methods of schemes 1-4 as described hereinbelow, wherein X and Y are independently selected from the group consisting of Se, S, O, NCH3 and NH.
The selenophene compounds of this invention are readily formulated into pharmaceutical compositions, also within the scope of this invention, for use in the presently described method for treatment of patients having tumors, In one preferred embodiment of this invention, the pharmaceutical composition comprises an anti-tumor effective amount of a selenophene compound of formula I: 
wherein R1 and R2 are independently selected from the group consisting of; 
H, CHO, CH2OH and CH2NH2,
X and Y are independently selected from the group consisting of Se, S, O and NR, wherein R is H or C1-C7 alkyl
R3, R4, R5 and R6 are independently selected from the group consisting of nitro, amino, alkoxy, cyano, chloro, bromo, iodo, C1-C7 alkyl or haloalkyl, C1-C7 alkenyl or haloalkenyl, C1-C4 alkanoyloxy methyl, CH2OR7, COR8, CH2NR9R10, CH(OR7)R11, CHxe2x95x90CR12R13, CHxe2x95x90NR14, CH2SC(NH)NH2 and Cxe2x89xa1CR15 wherein
R7 is H, CO(CH2)2CO2H, (CH2)2OCH3, C1-C4 alkyl or COC1-C17 alkyl;
R8 is H or C1-C7 alkyl;
R9 and R10 are independently H, CN, C1-C4 alkyl, or mono- or di-hydroxyC2-C4 alkyl;
R11 is C1-C7 alkyl, or C1-C7 alkenyl;
R12 and R13 are independently H, C1-C7 alkyl, COOR8, CN, CH(OR7)COOR8, Br, CO-thienyl, COC6H4OH(p);
R14 is NR7 or OR8;
R15 is COOR8, CH(OR7)CH2OR16 or CH(OCOC1-C4 alkyl)CH2OR8;
R16 is H, COCH2CH2CO2H, or COC1-C7 alkyl;
cyclodextrin complexes of such compound and when R3, R4, R5 or R6 is CH2NR6R7, the pharmaceutically acceptable salt of the compound represented thereby, and a pharmaceutically acceptable carrier.
Another pharmaceutical composition within the scope of this invention comprises an anti-tumor effective amount of a selenophene compound of the formula I: 
wherein R1 and R2 are independently selected from the group consisting of; 
H, CHO, CH2OH and CH2NH2;
X and Y are independently selected from the group consisting of Se, S, O NCH3 and NH;
R3, R4 and R6 are H;
R5 is selected from the group consisting of H, CHO, CH2OH and CH2NH2; cyclodextrin complexes of such compounds; and when R3, R4, R5 or R6 is CH2NH2, the pharmaceutically acceptable salt of the compound represented thereby; with the proviso, that when R2 is 
and a pharmaceutically acceptable carrier.
The present compounds are readily prepared using art-recognized chemical-synthesis procedures as exemplified hereinbelow.
The cytotoxic activity of the present selenophene compounds have been measured utilizing two different assays or screens. The first screen measures the cytotoxicity against a panel of sixty different human tumor cell lines. This assay provides data regarding the general cytotoxicity of an individual compound. In particular this type of assay is useful in identifying compounds which have enhanced cytotoxic activity against slow growing tumors as compared to faster growing tumor cells such as leukemia tumor cell lines. The identification of such compounds is critical since previously identified antitumor agents have low cytotoxic activity against slower growing tumors. The specificity of a compound for a limited number of tumor cell lines also indicates that such a compound will likely be less cytotoxic to normal cells. The specificity of a cytotoxic compound for tumor cell lines relative to normal cells is an important characteristic of an effective antitumor agent.
Antitumor cytotoxicity data generated from the National Cancer Institute human tumor cell panels can also be expressed in a graphic pattern (mean graph) to display differential cell growth inhibition (K. D. Paull, R. H. Shoemaker, L. Hodes, A. Monks, D. A. Scudiero, L. Rubinstein, J. Plowman and M. R. Boyd, J. Natl. Cancer Inst., 81, 1088, 1989.) In the mean graph, the arithmetic mean of the logarithm of the GI50 (50% growth inhibition), TGI (total growth inhibition) or LC50 (50% lethal concentration) values is used as an anchor point. Relative cytotoxicity is displayed by projecting bars to the right or left of the mean, depending on whether cell sensitivity to a test compound is more or less than average. The length of a bar is indicative of differential cytotoxicity against a specific type of tumor cells or tumor panels.
In a second assay, the cytotoxic selectivity is assessed by comparing compound cytotoxicity against human renal carcinoma cells (A-498), ras-transformed human bronchial epithelial cells (TBE) and normal human renal cells (RPTEC). IC50 values were compared between treated TBE cells and RPTEC cells and the selective cytotoxicity index (SCI) was determined [SCI=GI50(RPTEC)/GI50 (A-498)].
The antitumor cytotoxicity of the selenophene compounds tested in those in vitro assays was measured by a microculture assay using either a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) or sulforhodamine B (SRB) based assay. [M. R. Boyd in xe2x80x9cPrinciples and Practices of Oncology,xe2x80x9d V. T. DeVita, Jr.]. The experiments were conducted at Purdue University in 96-well microtiter plates and the cytotoxic effects of the selenophene compounds on those cells were measured by cell count using a Coulter Z. F. counter (Hialeah, Fla.). The results are expressed as GI50, the concentration of drug at which cell numbers are reduced to 50% of control cell culture [T. C. K. Chan, C. J. Chang, N. M. Koonchanok and R. L. Geahlen, Biochem. Biophys. Res. Commun., 193, 1152, (1993); S. Hellman and S. A. Rosenberg (Eds.), Vol. 3, PPO Updates, Number 10, (1989).]
This in vitro microculture assay has an advantage over in vivo assays in that results are obtained within a week as opposed to several months. The MTT assay is based on the production of a dark blue formazan product by dehydrogenase in the mitochondria of live tumor cells after exposure to drug for 6 days [M. C. Alley, D. A. Scudiero, A. Monks, M. L. Hursey, M. J. Czerwinski, D. L. Fine, B. J. Abbott, J. G. Mayo, R. H. Shoemaker and M. R. Boyd, Cancer Res., 48, 5 89, 1988]. Thus, only live cells are stained and can be measured at 570 nm. The SRB assay is based on the binding of the anionic group to the basic amino acid residues of cellular proteins after exposure of tumor cells to drug for 2 days [ P. Skehan, R. Storeng, D. Scudiero, A. Monks, J. McMahon, D. Vistica, J. T. Warren, H. Bohesch, S. Kenney and M. R. Boyd, J. Nat. Cancer Inst., 82, 1107, 1990.] Thus, the total protein can be measured at 564 nm. Antitumor cytotoxicity is reported as GI50, effect drug dose at which cell growth is retarded to 50% of control culture of tumor cells. Thee active compounds are defined as those compounds having GI50 values that are less than 10xe2x88x924 M or 10 xcexcg/ml.
The data presented in Table 1 illustrates that selenophenes generally exhibit greater cytotoxicity for human renal carcinoma cells in comparison to the normal human cells. The data of Table 1 demonstrates the selective cytotoxicity of various selenophene compounds against human renal carcinoma and ras-oncogene transformed human bronchial epithelial cells [in GI50(ug/ml)]. The following abbreviations are used for the tested cell lines:
RPTEC: normal human renal cells
A-498: human renal carcinoma
TBE: ras-transformed human bronchial epithelial cells
SCI: selectively cytotoxicity index=GI50 (RPTEC)/GI50) (A-498)
The present invention further provides pharmaceutical formulations comprising an effective amount of a selenophene compound for treating a patient having a tumor. As used herein, an effective amount of the selenophene compound is defined as the amount of the compound which, upon administration to a patient, inhibits growth of tumor cells, Kills malignant cells, reduces the volume or size of the tumors or eliminates the tumor entirely in the treated patient.
The effective amount to be administered to a patient is typically based on body surface area, patient weight, and patient condition. The interrelationship of dosages for animals and humans (based on milligrams per meter squared of body surface) is described by Freireich, E. J., et al., Cancer Chemother. Rep., 50 (4): 219 (1966). Body surface area may be approximately determined from patient height and weight (see e.g., Scientific Tables, Geigy Pharmaceuticals, Ardley, N.Y., pages 537-538 (1970)). An effective amount of the selenophene compounds in the present invention can range from about 5 mg/kg to about 100 mg/kg, more preferably from about 0.25 mg/kg to about 50 mg/kg, and most preferably about 0.1 to about 10 mg/kg.
Effective doses will also vary, as recognized by those skilled in the art, dependant on route of administration, excipient usage and the possibility of co-usage with other therapeutic treatments including other anti-tumor agents, and radiation therapy.
The pharmaceutical formulation may be administered via the parenteral route, including subcutaneously, intraperitoneally, intramuscularly and intravenously. Examples of parenteral dosage forms include aqueous solutions of the active agent, in a isotonic saline, 5% glucose or other well-known pharmaceutically acceptable liquid carrier. In one preferred aspect of the present embodiment, the selenophene compound is dissolved in a saline solution containing 5% of dimethyl sulfoxide and 10% Cremphor EL (Sigma Chemical Company). Additional solubilizing agents such as cyclodextrins, which form specific, more soluble complexes with the present selenophene compounds, or other solubilizing agents well-known to those familiar with the art, can be utilized as pharmaceutical excipients for delivery of the selenophene compounds.
The present compound can also be formulated into dosage forms for other routes of administration utilizing well-known methods. The pharmaceutical compositions can be formulated, for example, in dosage forms for oral administration in a capsule, a gel seal or a tablet. Capsules may comprise any well-known phannaceutically acceptable material such as gelatin or cellulose derivatives. Tablets may be formulated in accordance with conventional procedure by compressing mixtures of the active polythiophene and solid carriers, and lubricants well-known to those familiar with the art. Examples of solid carriers include starch, sugar, bentonite. The compounds of the present invention can also be administered in a form of a hard shell tablet or capsule containing, for example, lactose or mannitol as a binder and conventional fillers and tableting agents.
The following examples are provided to illustrate various embodiments of Applicants"" invention, and are not intended to in any way limit the scope of the invention as set forth in this specification and appended claims.